Project 1: Epigenetic control of neurogenesis in different hESC lines Human embryonic stem cells (hESCs) have garnered tremendous public interest for their promising selfrenewal capacities and differentiation potentials which are ideal for tissue repair. However, the many available hESC lines were established using embryos with different human genetic backgrounds and with widely varying culturing procedures. Such differences could have a huge influence on the genetic stability, epigenetic, and ultimately cellular properties of hESCs, and therefore influence the usage of these cells in regenerative medicine. To begin to characterize the differences between the various hESC lines, we have shown that while one of the hESC lines (the HSF1 line) produces neurons primarily of forebrain origin, the other (HSF6) primarily generates neurons of mid-/hind-brain and spinal cord origins with GABAergic, glutamatergic, dopaminergic, seratonergic, and cholinergic neuretransmitter traits. These observations suggest that these two hESC lines already have lineage differentiation bias even at the ESC stage. Here we propose to study the nature of the potential epigenetic pre-programing events among different hESC lines with three specific aims: Aim 1, To determine the neuronal subtype differentiation properties of four independent hESC lines, namely H1 and H9 in addition to HSF1 and HSF6; Aim 2, To employ mRNA as well as microRNA expression array analyses, quantitative RT-PCR, and Western blotting methods to evaluate the differences in gene expression profiles between these four hESC lines, both at the ES cell stage and after ES cells are converted into neural stem/progenitor cells as well as post-mitotic neurons; and Aim 3, To examine whether these four different hESC lines differ in their genomes and epigenomes (genome-wide DMA methylation and histone modification patterns) at ESC and NPC stages. By defining the mechanisms by which these commmonly used hESC lines preferentially diferentiate into different subtypes of neurons, our studies will set the stage and create methods for future characterization of additional hESC lines to assess their differentiation potentials/bias, which will be extremely valuable for any future use of hESCs in regenerative medicine, inlcuding neural stem cell therapy and the establishment of novel neurological disease models using hESC-derived neurons since regional specificity is key to many neurological disorders.